O 2-(N -Hydroxy(methoxy)-2-ethanesulfonamido) protected diazen-1-ium-1,2-diolates: Nitric oxide release via a base-induced β-elimination cleavage

Article abstract of DOI:10.1021/ol200053z

O²-(Ethanesulfohydroxamic acid) and O²-(N-methoxy-2- ethanesulfonylamido) diazen-1-ium-1,2-diolates (4-7), a novel type of O ²-(protected) diazeniumdiolate, were synthesized using a key thioacetate oxidation reaction. Nitr

Full text of DOI:10.1021/ol200053z

ORGANIC
LETTERS
2
O -(N-Hydroxy(methoxy)-2-
ethanesulfonamido) Protected Diazen-1-
ium-1,2-diolates: Nitric Oxide Release via
a Base-Induced β-Elimination Cleavage
2
011
Vol. 13, No. 5
1178–1181
Zhangjian Huang and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton,
Alberta, T6G 2N8, Canada
eknaus@pharmacy.ualberta.ca
Received January 7, 2011
ABSTRACT
2
2
2
O -(Ethanesulfohydroxamic acid) and O -(N-methoxy-2-ethanesulfonylamido) diazen-1-ium-1,2-diolates (4-7), a novel type of O -(protected)
diazeniumdiolate, were synthesized using a key thioacetate oxidation reaction. Nitric oxide release studies showed that O -(N-methoxy-2-
2
ethanesulfonylamido) diazeniumdiolates 5 and 7 released NO in a nonphysiological alkaline buffer, in the presence of bases such as the basic
natural amino acids Arg and His, or the non-nucleophilic organic base DBU in PBS at pH 7.4, via a β-elimination cleavage reaction.
2
include O -vinyl, O -acetoxymethyl, O -glycosylated,
6
2
7
2
8
Diazeniumdiolates, which can spontaneously release
nitric oxide (NO) under physiological conditions (pH
2
and O -(2,4-dinitrophenyl) diazeniumdiolates. These
9
2
O -protected compounds, which are metabolized by en-
7
to several days, display vasorelaxant, antithrombotic,
.4, 37 °C) with a range of half-lives from a few seconds
1
2
3
6
zymes such as hepatic cytochrome P450, esterase, certain
7
4
cytostatic, and genotoxic activities. Selective protection
5
8
glycosidases, and glutathione/glutathione S-transferase
2
at the terminal oxygen atom (O ) of the diazen-1-ium-1,2-
10
(GSH/GST), have been studied extensively. Although
there have been many attempts directed toward the devel-
diolate offers a method to facilitate delivery of the NO-
donor to the target tissue site and release of NO to induce
2
the desired therapeutic effect. A number of different O -
protected diazeniumdiolates have been reported that
2
opment of O -protected diazeniumdiolates as potential ther-
apeutic candidates, no compound has achieved clinical
approval to date. As part of our ongoing program to
investigate the application of NO-donors as potential
2
(
1) (a) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A.
therapeutic agents, we now report a new type of O -ethane-
2
sulfohydroxamic acid and O -(N-methoxy-2-ethanesulfony-
Methods Enzymol. 1996, 268, 281–293. (b) Fitzhugh, A. L.; Keefer, L. K.
Free Radical Biol. Med. 2000, 28, 1463–1469.
(
2) Morley, D.; Maragos, C. M.; Zhang, X. Y.; Boignon, M.; Wink,
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8) Wu, X.; Tang, X.; Xian, M.; Wang, P. G. Tetrahedron Lett. 2001,
2, 3779–3782.
lamido) protected diazen-1-ium-1,2-diolates. These com-
pounds are NO donors which can release NO in the presence
of a base, including the basic natural amino acids Arg and
His, via a β-elimination cleavage reaction.
(
(
(
(9) Saavedra, J. E.; Srinivasan, A.; Bonifant, C. L.; Chu, J.; Shanklin,
A. P.; Flippen-Anderson, J. L.; Rice, W. G.; Turpin, J. A.; Davies,
K. M.; Keefer, L. K. J. Org. Chem. 2001, 66, 3090–3098.
(10) Shami, P. J.; Saavedra, J. E.; Wang, L. Y.; Bonifant, C. L.;
Diwan, B. A.; Singh, S. V.; Gu, Y.; Fox, S. D.; Buzard, G. S.; Citro,
M. L.; Waterhouse, D. J.; Davies, K. M.; Ji, X.; Keefer, L. K. Mol.
Cancer Ther. 2003, 2, 409–417.
(
(
(
4
1
0.1021/ol200053z r 2011 American Chemical Society
Published on Web 02/09/2011

are useful model compounds to study the potential
β-elimination reaction in the absence of NO release. We
now describe the synthesis, β-elimination, and NO release
2
properties for the O -(ethanesulfohydroxamic acid) and
2
O -(N-methoxy-2-ethanesulfonylamido) derivatives of the
pyrrolidine and prolinol diazen-1-ium-1,2-diolate com-
pounds 4-7 illustrated in Figure 1.
Scheme 1. Synthesis of the Pyrrolidine Analogs 4 and 5
Figure 1. Rationale for the design of novel base-sensitive
O -(ethanesulfonyl) diazen-1-ium-1,2-diolates.
2
2
The concept that O -(ethanesulfonyl) diazen-1-ium-1,2-
diolates will undergo a base-induced β-elimination reac-
tion is based on the knowledge that (i) 2-chloroethanesul-
fonyl chloride (1), upon reaction with an amine, is reported
to release HCl (dehydrochlorination) to form a vinylsul-
1
1
According to previous reports, it is likely that the
PYRRO/NO compound 11 could act as a base to induce
β-elimination (-HCl) from a 2-chloroenthanesulfonyl
compound to give a vinyl product and release of NO from
the PYRRO/NO reactant 11, rather than condensation
1
1
fonylamide product 2 (Figure 1) and (ii) the N-methox-
yethanesulfonylamide ester of ibuprofen (3), which was
developed by our group and shown to exhibit more potent
anti-inflammatory activity (AI) than the parent drug
1
2
2
ibuprofen, released ibuprofen in the presence of the
non-nucleophilic base 1,8-diazabicyclo[5.4.0]undec-7-ene
with the chloro compound to form the desired O -alkylation
product. Consequently, an alternative synthetic strategy
was required. It is reported that oxidation of a thioacetate
using aqueous hydrogen peroxide (H O ) and sodium
(
DBU) (Figure 1). Accordingly, it was of interest to
investigate whether coupling a diazen-1-ium-1,2-diolate
NONOate) moiety to the β-position of an ethanesulfonyl
2
2
13
(
acetate affords a sodium sulfonate. It was anticipated that
2
2
group would provide a hitherto-unknown class of O -
protected diazen-1-ium-1,2-diaolates in which the NON-
Oate moiety acts initially as a leaving group via a base-
induced β-elimination which would subsequently release
NO. Credence for this objective is consistent with previous
data showing that (i) alkylsulfohydroxamic acids release
NO in PBS at pH 7.4, and nitroxyl (HNO) in the presence
β-elimination would not occur when O -alkylation of a
diazen-1-ium-1,2-diolate was performed since the thioace-
tate group is a weak electron-withdrawing moiety compared
to a sulfohydroxamic group. The synthetic methods used to
prepare the target sulfohydroxamic products 4 and 5 are
depicted in Scheme 1. Thus, reaction of 1,2-dibromoethane
8
2
9
with potassium thioacetate furnished the monosubstituted
-bromoethyl thioacetate (9). The 2-bromoethyl compound
was transformed by reaction with sodium iodide in
1
2
of base, to act as NO/HNO donors and (ii) N-methox-
ysulfonylamides, which cannot act as NO/HNO donors,
acetone to the more reactive 2-iodoethyl thioacetate (10)
2
to improve the yield in the subsequent O -alkylation reac-
2
tion. In this regard, O -alkylation of PYRRO/NO 11 using
(
11) (a) Davies, W. G.; Hardisty, E. W.; Nevell, T. P.; Peters, R. H. J.
Chem. Soc. B 1970, 5, 998–1004. (b) Hromatka, O.; Binder, D.;
Knollmueller, M. Monatsh. Chem. 1968, 99, 1062–1067. (c) Johnson,
O. H.; Holum, J. R. J. Org. Chem. 1958, 23, 738–740.
(
12) Huang, Z.; Vel ꢀa zquez, C. A.; Abdellatif, K. A.; Chowdhury,
M. A.; Reisz, J. A.; DuMond, J. F.; King, S. B.; Knaus, E. E. J. Med.
Chem. 2010, dx.doi.org/10.1021/jm101403g.
(13) (a) Showell, J. S.; Russell, J. R.; Swern, D. J. Org. Chem. 1962,
27, 2853–2858. (b) Brouwer, A. J.; Ceylan, T.; Linden, T. V. D.;
Liskamp, R. M. Tetrahedron Lett. 2009, 50, 3391–3393.
Org. Lett., Vol. 13, No. 5, 2011
1179

2
0 furnished the O -(2-acetthioethyl) 1-(pyrrolidin-1-yl)-
1
diazen-1-ium-1,2-diolate (12) in 20% yield. Although the
NONO moiety is quite sensitive to an acidic medium,
furnished the sodium sulfonate 17 in 75% yield. Transfor-
mation of the sodium sulfonate 17 to the sulfonyl chloride by
reaction with thioyl chloride and the subsequent reaction of
sulfonyl chloride with hydroxylamine hydrochloride or
methoxylamine hydrochloride in the presence of potassium
carbonate or sodium bicarbonate in dry THF afforded the
respective target products 6 and 7.
2
fortunately the thioacetate moiety in the O -alkylated dia-
zeniumdiolate 12 can be converted to the sodium sulfonate
analog 13 in the presence of H O and sodium acetate in a
2
2
solution of acetic acid at 55 °C with a reaction time of 10 h in
a 66% yield without decomposition of the NONO moiety.
Reaction of the sodium sulfonate 13 with thionyl chloride in
DMF afforded the corresponding relatively unstable sulfo-
nyl chloride product which was used immediately in the
subsequent reaction without further purification. Thus,
reaction of this sulfonyl chloride with hydroxylamine hy-
drochloride in the presence of potassium carbonate in dry
THF afforded the target sulfohydroxamic product 4. A
similar reaction of the sulfonyl chloride with methoxylamine
hydrochloride in the presence of sodium bicarbonate in dry
THF furnished the N-methoxysulfonylamide product 5.
Table 1. Amount of Nitric Oxide (nmol) Released from 4-7, 11
a
(
120 nmol) in PBS at Different pH Values
PBS
PBS
PBS
PBS 7.4 þ
b
b
b
c
compd
4.0
7.4
10.0
Serum
4
5
6
7
0
86
0.74
94
10
0
43
8.6
1.6
2.6
;
3.4
3.1
;
36
1.1
41
26
11
223
;
a
-
The amount of NO
2
(nmol) arising from 120 nmol test compound.
formation is an equimolar reflection of the
O to produce nitrite. The result is the mean value
-
It is assumed that NO
reaction of NO with H
2
2
Scheme 2. Synthesis of the Prolinol Analogs 6 and 7
of three measurements (n = 3) where the variation from the mean
value was e2%; A solution of the test compound (120 nmol) in
b
phosphate buffer (2.4 mL) at various different pH values (4.0, 7.4, or
1
c
0.0) was incubated at 37 °C for 1.5 h. A solution of the test compound
120 nmol) in phosphate buffer (2.4 mL) at pH 7.4, to which 90 μL of rat
(
serum had been added, was incubated at 37 °C for 1.5 h.
2
The amount (nmol) of NO released from the O -[N-
hydroxy(methoxy)-2-ethanesulfonamido]diazen-1-ium-
1
,2-diolates 4-7 and PYRRO/NO 11 (120 nmol) upon
incubation in phosphate-buffered-solution (PBS) at pH
.0, PBS at pH 7.4, PBS at pH 10.0, or PBS at pH 7.4
4
containing rat serum were measured by quantitation of
nitrite using the Griess reaction (see data in Table 1).
Compounds 4 and 6 can theoretically release 3 equiv of
NO (1 equiv from the SO NHOH moiety and 2 equiv from
2
the diazeniumdiolate moiety). Compounds 5, 7, and 11 can
theoretically release 2 equiv of NO from the diazenium-
diolate moiety. In PBS pH 4.0, all target compounds
released a very low amount of NO (0-3.4 nmol of NO2-
were detected from 120 nmol of the test compound),
showing a good stability in an acidic medium. In PBS
pH 7.4 sulfohydroxamic acids 4 and 6 released more NO
relative to their O-methyl counterparts 5 and 7 (86 > 43
nmol, for 4 and 5, 36 > 26 nmol, for 6 and 7, respectively),
which can be attributed to the alkylsulfohydroxamic acid
1
2
Given that prolinol may have a more favorable toxico-
logical profile compared to pyrrolidine, the prolinol diaze-
niumdiolate analogues 6and 7were synthesized as illustrated
in Scheme 2. Condensation of sodium 1-[2-hydroxymethyl]-
pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (14) with 2-iodo-
moiety acting as a NO donor in PBS at pH 7.4. It is
interesting that the N-methoxy compounds 5 and 7 release
moreNOatpH 10.0thanatpH 7.4 (94and 43nmol for 5 at
pH 10.0 and 7.4; 41 and 26 nmol for 7 at pH 10.0 and 7.4).
Since the N-methoxysulfonylamide moiety cannot act as a
NO donor, the higher NO release at pH 10.0 likely arises
from the NONO moiety after its base-induced β-elimina-
tion. Furthermore, in PBS at pH 10.0, NO release from the
sulfohydroxamic acids 4 and 6 was suppressed (0.74-1.1
nmol). One plausible explanation for this reduction in NO
release could be due to the fact that the sulfohydroxamic
acid moiety, at nonphysiological alkaline pH, decomposes
2
ethyl thioacetate (10) furnished the O -(2-acetthioethyl)
prolinoldiazeniumdiolate 15 in 24% yield. Since the hydro-
xyl group present in the prolinol derviative 15 will be
sensitive to thionyl chloride during a subsequent step to
prepare the sulfonyl chloride, this hydroxyl group was
acetylated using acetyl bromide to protect the hydroxyl
group as the acetate 16. Oxidation of the thioacetate moiety
present in 16 upon treatment with H O and sodium acetate
1
4
to release HNO (rather than NO) and sodium sulfinate
2
2
1
180
Org. Lett., Vol. 13, No. 5, 2011

the presence of basic natural amino acids [arginine (Arg),
histidine (His)] at physiological pH. Therefore, NO release
from 5 and 7 upon incubation in PBS at pH 7.4 containing
DBU, Arg, or His was investigated (see data in Table 2).
The amounts of NO2 (nmol) produced from 5 (120
nmol) in the presence of DBU, Arg, or His are 98, 96, and
Table 2. Amount of Nitric Oxide (nmol) Released from 5 and 7
120 nmol) in the Presence of Basic Compounds at Physiological
pH
(
a
-
PBS þ
PBS þ
PBS þ
H
2
O þ
d
b
b
b
c
compd
DBU
Arg
His
2
H O
DBU
89 nmol, respectively, which are similar to that arising from 5
5
7
98
34
96
34
89
29
36
;
209
;
in PBS at pH 10.0 (94 nmol, in Table 1). Similar comparisons
were observed for compound 7. Furthermore, the amounts of
NO released from 5 (120 nmol) in H O alone and in H O
a
-
2
2
The amount of NO
The result is the mean value of three measurements (n = 3) where
2
(nmol) arising from 120 nmol test compound.
containing DBU (240 nMol) were 36 and 209 nmol, respec-
tively. In this regard, the basic amino acids Arg and His, and
the organic non-nucleophilic base DBU, at physiological
pH appear to function similar to that observed at a non-
physiological alkaline pH of 10 with respect to inducing
b
variation from the mean value was e2%. A solution of the test
compound (120 nmol) in phosphate buffer (2.4 mL) at pH 7.4 containing
basic compound DBU, Arg, or His (240 nMol) was incubated at 37 °C
for 1.5 h. A solution of the test compound (120 nmol) in ultrapure water
2.4 mL) was incubated at 37 °C for 1.5 h. A solution of the test
compound (120 nmol) in ultrapure water (2.4 mL) containing DBU (240
nmol) was incubated at 37 °C for 1.5 h.
c
d
(
2
a β-elimination process for these O -((N-methoxy)ethane-
sulfonylamido) diazen-1-ium-1,2-diolates to free the NONO
moiety which then acts as a NO donor to release NO.
2
In summary, the stability data for the new O -(ethanesul-
fohydroxamic acid) protected diazeniumdiolates (4, 6) in the
presence of serum at physiological pH, and the capability of
2
the O -((N-methoxy)ethanesulfonylamido) diazeniumdio-
lates (5, 7) to release NO in the presence of basic compounds,
particularly basic amino acids, at physiological pH has been
acquired. These data provide insight into their relevant
pharmaceutical properties that are useful for developing
drug design strategies pertaining to NONOate donors.
which would prevent any subsequent β-elimination from
occurring that is necessary to release the NONO moiety.
On the other hand, NO release from 4-7 in PBS contain-
ing rat serum was suppressed (1.6-10 nmol) possibly due
to strong serum protein binidng, which is consistent with a
similar serum effect previously observed for aryl and alkyl
1
2,15
sulfohydroxamic acids.
These preliminary NO release studies indicate that
N-methoxysulfonylamides 5 and 7 can decompose to
release NO via a nonphysiological alkaline-induced β-eli-
mination process. It was also of interest to investigate
whether this β-elimination process could be induced in
Acknowledgment. We are also grateful to the Canadian
Institutes of Health Research (Grant No. MOP-14712) for
financial support of this research.
(
14) (a) Bonner, F. T.; Ko, Y. Inorg. Chem. 1992, 31, 2514–2519. (b)
Fukuto, J. M.; Hszieh, R.; Gulati, P.; Chiang, K. T.; Nagasawa, H. T.
Supporting Information Available. Experimental pro-
cedures and compound characterization for all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Biochem. Biophys. Res. Commun. 1992, 187, 1367–1373.
(
15) Huang, Z.; Vel ꢀa zquez, C. A.; Abdellatif, K. A.; Chowdhury,
M. A.; Jain, S.; Reisz, J. A.; DuMond, J. F.; King, S. B.; Knaus, E. E.
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Org. Lett., Vol. 13, No. 5, 2011
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